Synthesis and biochemical properties of cyanuric acid nucleoside- containing DNA oligomers

Article abstract of DOI:10.1021/tx980255e

1-(2-Deoxy-β-D-erythro-pentofuranosyl)cyanuric acid (cyanuric acid nucleoside, dY) (1) has been shown to be formed upon exposure of DNA components to ionizing radiation and excited photosensitizers. To investigate the biological and structural significance of dY residue in DNA, the latter modified 2'-deoxynucleoside was chemically prepared and then site- specifically incorporated into oligodeoxyribonucleotides (ODNs). This was achieved in good yields using the phosphoramidite approach. For this purpose, a convenient glycosylation method involving 3,5-protected 2- deoxyribofuranoside chloride and cyanuric acid (2,4,6-trihydroxy-1,3,5- triazine) was devised. The anomeric mixture of modified 2'- deoxyribonucleosides (1/2 α/β) was resolved by silica gel purification of the 5'-O-dimethoxytritylated derivatives, and then, phosphitylation afforded the desired β-phosphoramidite monomer (5). After solid-phase condensation and final deprotection, the purity and the integrity of the modified synthetic DNA fragments were checked using different complementary techniques such as HPLC and polyacrylamide gel electrophoresis, together with electrospray ionization and MALDI-TOF mass spectrometry. The presence of cyanuric acid nucleoside in a 14-mer was found to have destabilizing effects on the double-stranded DNA fragment as inferred from melting temperature measurements. The piperidine test applied to dY-containing ODNs supported the high stability of cyanuric acid nucleoside inserted into the oligonucleotide chain. Several enzymatic experiments aimed at determining the biological features of such a DNA lesion were carried out. Thus, processing of dY by nuclease P₁, snake venom phosphodiesterase (SVPDE), calf spleen phosphodiesterase (CSPDE), and repair enzymes, including Escherichia coli endonuclease III (endo III) and Fapy glycosylase (Fpg), was investigated. Finally, a 22-mer ODN bearing a cyanuric acid residue was used as a template to study the in vitro nucleotide incorporation opposite the damage by the Klenow fragment of E. coli polymerase I.

Full text of DOI:10.1021/tx980255e

630
Chem. Res. Toxicol. 1999, 12, 630-638
Syn th esis a n d Bioch em ica l P r op er ties of Cya n u r ic Acid
Nu cleosid e-Con ta in in g DNA Oligom er s
Didier Gasparutto,† Sandrine Da Cruz,† Anne-Gae¨lle Bourdat,†
Michel J aquinod,‡ and J ean Cadet*,†
Laboratoire des Le´sions des Acides Nucle´iques, Service de Chimie Inorganique et Biologique,
De´partement de Recherche Fondamentale sur la Matie`re Condense´e, CEA-Grenoble,
F-38054 Grenoble Cedex 9, France, and Laboratoire de Spectrome´trie de Masse des Prote´ines,
Institut de Biologie Structurale, F-38027 Grenoble Cedex, France
Received November 23, 1998
1-(2-Deoxy-â-D-erythro-pentofuranosyl)cyanuric acid (cyanuric acid nucleoside, dY) (1) has
been shown to be formed upon exposure of DNA components to ionizing radiation and excited
photosensitizers. To investigate the biological and structural significance of dY residue in DNA,
the latter modified 2′-deoxynucleoside was chemically prepared and then site-specifically
incorporated into oligodeoxyribonucleotides (ODNs). This was achieved in good yields using
the phosphoramidite approach. For this purpose, a convenient glycosylation method involving
3,5-protected 2-deoxyribofuranoside chloride and cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine)
was devised. The anomeric mixture of modified 2′-deoxyribonucleosides (1/2 R/â) was resolved
by silica gel purification of the 5′-O-dimethoxytritylated derivatives, and then, phosphitylation
afforded the desired â-phosphoramidite monomer (5). After solid-phase condensation and final
deprotection, the purity and the integrity of the modified synthetic DNA fragments were checked
using different complementary techniques such as HPLC and polyacrylamide gel electrophore-
sis, together with electrospray ionization and MALDI-TOF mass spectrometry. The presence
of cyanuric acid nucleoside in a 14-mer was found to have destabilizing effects on the double-
stranded DNA fragment as inferred from melting temperature measurements. The piperidine
test applied to dY-containing ODNs supported the high stability of cyanuric acid nucleoside
inserted into the oligonucleotide chain. Several enzymatic experiments aimed at determining
the biological features of such a DNA lesion were carried out. Thus, processing of dY by nuclease
P1, snake venom phosphodiesterase (SVPDE), calf spleen phosphodiesterase (CSPDE), and
repair enzymes, including Escherichia coli endonuclease III (endo III) and Fapy glycosylase
(Fpg), was investigated. Finally, a 22-mer ODN bearing a cyanuric acid residue was used as
a template to study the in vitro nucleotide incorporation opposite the damage by the Klenow
fragment of E. coli polymerase I.
xenobiotics, etc.) agents (6, 7). Several studies have
shown that 8-oxodGuo, which exhibits a lower ionization
potential than the normal DNA nucleosides, is highly
reactive toward oxidizing reagents (8-11). This suggests
that the latter nucleoside may also be sensitive to in vivo
DNA oxidation. Recently, 8-oxodGuo has been found to
be an efficient substrate of photosensitized reactions
which give rise to a set of secondary products. Thus, at
the nucleoside level, it was shown that the major product
of singlet oxygen oxidation (type II mechanism) was the
cyanuric acid nucleoside (1) (12, 13). Although the latter
model studies provided a large amount of information
about the structure and the mechanism of formation of
1, the presence of dY has not been established so far at
the DNA level.
In this paper, we report the first synthesis of cyanuric
acid nucleoside (1) and its site-specific insertion into
several oligodeoxyribonucleotides using phosphoramidite
chemistry. The modified DNA oligomers were used to
determine the hybridization properties of 1 upon substi-
tution of the guanine parent base. In addition, informa-
tion about biochemical features, including enzymatic
cleavage, repair, and mutagenicity, is provided.
In tr od u ction
Several studies have established that 8-oxo-7,8-dihy-
dro-2′-deoxyguanosine (8-oxodGuo),1 which may result
from hydroxylation of the C8 position of 2′-deoxygua-
nosine (dGuo), is the major stable modified nucleoside
present in oxidized DNA. The structural and biological
features of 8-oxodGuo have been widely studied during
the past decade (1-5). Interestingly, 8-oxodGuo is cur-
rently used as a biomarker of DNA oxidation induced by
both endogenous (cellular metabolism, oxidative stress,
etc.) and exogenous (ionizing radiation, photosensitizers,
* To whom the correspondence should be addressed. Tele-
phone: (33)-4-76-88-49-87. Fax: (33)-4-76-88-50-90. E-mail: cadet@
drfmc.ceng.cea.fr.
† CEA-Grenoble.
‡ Institut de Biologie Structurale.
1 Abbreviations: dY, 1-(2-deoxy-â-D-erythro-pentofuranosyl)cyanuric
acid; 8-oxodGuo, 8-oxo-7,8-dihydro-2′-deoxyguanosine; ODN, oligode-
oxyribonucleotide; DMTrCl, 4,4′-dimethoxytrityl chloride; TFA, tri-
fluoroacetic acid; CSPDE, calf spleen phosphodiesterase; SVPDE, snake
venom phosphodiesterase; FAB-MS, fast atom bombardment mass
spectrometry; ESI-MS, electrospray ionization mass spectrometry;
MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-
flight mass spectrometry; PAGE, polyacrylamide gel electrophoresis;
endo III, endonuclease III; Fpg, formamidopyrimidine DNA glycosylase.
10.1021/tx980255e CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/25/1999

Oligonucleotides Containing Cyanuric Acid Nucleoside
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 631
Syn th etic P r oced u r es. (1) 1-(2-Deoxy-r,â-D-er yth r o-p en -
tofu r a n osyl)cya n u r ic Acid (1 a n d 2). A mixture of 2,4,6-
trihydroxy-1,3,5-triazine (330 mg, 2 mmol), 2-deoxy-3,5-di-O-p-
toluoyl-D-erythro-pentofuranosyl chloride (14) (800 mg, 2 mmol),
potassium nonafluorobutanesulfonate (1625 g, 2.4 equiv), hex-
amethyldisilazane (0.295 mL, 0.8 equiv), and trimethylsilyl
chloride (0.785 mL, 3.1 equiv) was refluxed in 20 mL of
anhydrous acetonitrile for 16 h, under conditions of moisture
exclusion. The course of the reaction was followed by TLC
(CHCl3/MeOH, 90/10, v/v). After evaporation of the solvents to
dryness, the residue was taken up in CHCl3 and washed
successively with saturated NaHCO3 and water. The major
products corresponding to the mixture of R and â anomers of
the 3′,5′-diprotected nucleoside (Rf ) 0.48) were not isolated,
but directly submitted to alkaline deprotection. Thus, the
glycosylation mixture was placed in a 1% NaOH methanol
solution (50 mL) which was stirred for 1 h at room temperature.
Solvents were removed by evaporation, and the resulting residue
was dissolved in water (50 mL), prior to being washed with
diethyl ether (50 mL) and chloroform (50 mL). The two anomers
1 and 2 were then purified on a short silica gel column that
was eluted with a step gradient of MeOH in CHCl3 (from 0 to
20%): yield of 270 mg (55%); 1/2 R/â (from integration of the
HPLC and NMR peaks area of the anomeric mixture); Rf ) 0.53
(70/30 CHCl3/MeOH); λmax (H2O, pH 7) 220 nm (shoulder);
ESI-MS (negative mode) m/z 244.22 [M - H]- (calcd mass
245.19).
For â anomer 1: 1H NMR (400.13 MHz, D2O) δ 11.39 (s, 2H,
NH), 6.64 (dd, J ) 5.3 and 8.6 Hz, 1H, H-1′), 4.63 (m, 1H, H-3′),
4.02 (m, 1H, H-4′), 3.93 (m, 1H, H-5′), 3.85 (m, 1H, H-5′′), 2.96
(m, 1H, H-2′), 2.35 (m, 1H, H-2′′); 13C NMR (100.61 MHz,
DMSO-d6) δ 149.50 (2C, C-2 and C-6), 148.70 (1C, C-4), 87.59
(1C, C-4′), 82.04 (1C, C-1′), 71.09 (1C, C-3′), 62.46 (1C, C-5′),
36.85 (1C, C-2′).
For R anomer 2: 1H NMR (400.13 MHz, D2O) δ 11.39 (s, 2H,
NH), 6.55 (pseudo-t, J ) 7.6 and 7.5 Hz, 1H, H-1′), 4.43 (m, 1H,
H-3′), 4.40 (m, 1H, H-4′), 3.91 (m, 1H, H-5′), 3.77 (m, 1H, H-5′′),
2.77 (m, 1H, H-2′), 2.72 (m, 1H, H-2′′); 13C NMR (100.61 MHz,
DMSO-d6) δ 149.69 (2C, C-2 and C-6), 148.70 (1C, C-4), 85.63
(1C, C-4′), 81.74 (1C, C-1′), 70.40 (1C, C-3′), 61.50 (1C, C-5′),
36.32 (1C, C-2′).
Ma ter ia ls a n d Meth od s
Gen er a l P r oced u r es a n d Ma ter ia ls. 2,4,6-Trihydroxy-
1,3,5-triazine (cyanuric acid) was from Fluka (Buchs, Switzer-
land). The silica gel (70-200 µm) used for the low-pressure
column chromatography was purchased from SDS (Peypin,
France). TLC was carried out on Merck DC Kieselgel 60 F-254
plastic sheets (Darmstadt, Germany). Acetonitrile and methanol
(HPLC grade) were obtained from Carlo Erba (Milan, Italy).
Buffers for high-performance liquid chromatography (HPLC)
were prepared using water purified with a Milli-Q system
(Milford, MA). The porous graphitized Hypercarb carbon column
(98.5% carbon, particle size of 5 µm, porosity of 250 Å, 100 mm
× 3 mm i.d.) was from Shandon (Runcorn, Cheschire, U.K.),
whereas the Hypersil ODS column (5 µm, 4.6 mm × 250 mm
i.d.) was purchased from Interchim (Montluc¸on, France). Un-
modified deoxyribonucleoside phosphoramidites, protected
with phenoxyacetyl for dAdo, isopropylphenoxyacetyl for dGuo,
and acetyl for dCyd, were from Glen Research (Sterling, VA).
Functionalized CPG supports, involving the phenoxyacetyl
protective group for dAdo and dGuo and the isobutyryl pro-
tective group for dCyd, were purchased from Sigma (St. Louis,
MO). 8-OxodGuo-containing oligonucleotide was prepared
using a commercial phosphoramidite monomer from Glen
Research.
En zym es. Calf spleen phosphodiesterase (CSPDE) and snake
venom phophodiesterase (SVPDE) were purchased from Boe-
hringer Mannheim (Mannheim, Germany). Fpg and endonu-
clease III were kind gifts from S. Boiteux (CEA, Fontenay-aux-
Roses, France). Nuclease P1 (Penicillium citrium) and phospha-
tase alkaline were purchased from Sigma. T4 polynucleotide
kinase was obtained from Pharmacia Biotech (Uppsala, Swe-
den). Klenow fragment (exo-) of Escherichia coli DNA poly-
merase I was from Amersham (Buckinghamshire, U.K.).
Ma ss Sp ectr om etr y Mea su r em en ts. FAB (fast atom bom-
bardment) mass spectrometry analyses were carried out on a
VG ZAB 2-EQ apparatus (Manchester, U.K.). The samples were
dissolved in either a glycerol or NBA matrix prior to be analyzed.
All modified and unmodified oligonucleotides were character-
ized by electrospray ionization mass spectrometry measure-
ments (ESI-MS) on a Micromass Platform model 3000 spectro-
photometer (Manchester, U.K.). Typically, 0.1 OD of the sample
was dissolved in a solution of acetonitrile and water (50/50, v/v)
containing 1% triethylamine prior to be analyzed in the negative
mode. The modified nucleoside dY and its 5′-DMTr-protected
derivative were analyzed by ESI-MS in both the negative and
positive modes. For the positive mode analysis, the sample was
dissolved in a solution of acetonitrile and water (50/50, v/v) that
contained 0.5% formic acid.
MALDI mass spectra were obtained with a commercially
available time-of-flight mass spectrometer (Voyager-DE, Per-
septive Biosystems, Framingham, MA) equipped with a 337 nm
nitrogen laser and a pulsed delay source extraction. Spectra
were recorded from 256 laser shots with an accelerating voltage
of 25 kV in the linear and positive modes. For the matrix, a
mixture of 3-hydroxypicolinic acid and picolinic acid in a 4/1
(w/w) ratio was dissolved in an aqueous acetonitrile solution
(50%) that contained 0.1% TFA and a small amount of Dowex-
50W 50X8-200 cation-exchange resin (Sigma). Typically, 1 µL
of the sample was added to 1 µL of the matrix, and the resulting
solution was stirred. The resulting sample was then placed on
the top of the target plate and allowed to dry by itself. The
spectra were calibrated with a 1 pmol/µL solution of myoglobin
(m/z 16 952), using the same conditions that were described for
the analysis of oligonucleotides.
La belin g of Oligon u cleotid es. Oligonucleotides (10 pmol)
were labeled at the 5′-terminus with 10 µCi of [γ-32P]ATP (2
pmol, 10 mCi/mL) from Amersham upon incubation with T4
polynucleotide kinase (9.5 units) in 10 µL of supplied buffer at
37 °C for 30 min. The reaction was quenched by addition of 1
µL of a 0.5 M EDTA solution (pH 8). Unincorporated [γ-32P]-
ATP was removed by purification of the oligonucleotide on a
MicroSpin column (Pharmacia Biotech).
(2) 1-[2-Deoxy-5-(4,4′-dim eth oxytr ityl)-r,â-D-er yth r o-p en -
tofu r a n osyl]cya n u r ic Acid (3 a n d 4). The anomeric mixture
of compounds 1 and 2 (0.41 mmol, 100 mg) was dried by
repeated coevaporation with anhydrous pyridine and then
dissolved in pyridine (10 mL). The solution was cooled in an
ice/water bath and kept under an argon atmosphere. Then, 4,4′-
dimethoxytrityl chloride (DMTrCl, 195 mg, 1.4 equiv) was added
under stirring. After 1 h, the cooling bath was removed and the
reaction was continued at room temperature overnight. Subse-
quently, 1 mL of methanol was added and the resulting solvent
mixture was evaporated. The oily residue was dissolved in ethyl
acetate (50 mL) and washed with a saturated NaHCO3 aqueous
solution (50 mL) and water (50 mL). The organic phase that
contained the two 5′-protected anomers 3 and 4 was dried over
Na2SO4 and evaporated under reduced pressure. The mixture
of R and â anomers was resolved by silica gel column chroma-
tography (0 to 5% step gradient of methanol in CHCl3 in the
presence of 1% triethylamine) to afford the title compounds 3
and 4 in a yield of 40.5 and 19%, respectively (1/2 R/â). One-
dimensional TLC analysis was performed using CHCl3/MeOH
(90/10, v/v) with 1% TEA as the solvent. The Rf values were
0.63 for the R-anomer 4 and 0.52 for the â-anomer 3: ESI-MS
(negative mode) m/z 546.09 [M - H]- (calcd mass 547).
For â anomer 3 (91 mg): 1H NMR (400.13 MHz, acetone-d6)
δ 7.62-6.85 (m, 12H, aromatic H of DMTr), 6.56 (dd, J ) 4.6
and 8.9 Hz, 1H, H-1′), 4.49 (m, 1H, H-3′), 3.98 (m, 1H, H-4′),
3.81 (s, 6H, OCH3), 3.39 (m, 1H, H-5′), 3.23 (m, 1H, H-5′′), 2.76
(m, 1H, H-2′), 2.21 (m, 1H, H-2′′).
For R anomer 4 (43 mg): 1H NMR (400.13 MHz, acetone-d6)
δ 7.60-6.78 (m, 12H, aromatic H of DMTr), 6.49 (pseudo-t, 1H,
H-1′), 4.44 (m, 1H, H-3′), 4.42 (m, 1H, H-4′), 3.80 (s, 6H, OCH3),

632 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Gasparutto et al.
3.36 (m, 1H, H-5′), 3.21 (m, 1H, H-5′′), 2.63 (m, 1H, H-2′), 2.57
(m, 1H, H-2′′).
with a freshly made 1 M piperidine aqueous solution at 90 °C
for 30 min. The reactions were carried out on 0.01 OD of 5′-
32P-labeled modified oligodeoxyribonucleotides in 100 µL of the
piperidine solution in sealed tubes. After cooling, the samples
were coevaporated twice with water and then loaded onto a 20%
polyacrylamide denaturing gel. The electrophoresis was carried
out at 1300 V for 3 h, and subsequently, the gel was exposed to
X-ray films.
Digestion of Mod ified ODNs by Nu clea se P 1 a n d Alk a -
lin e P h osp h a ta se. The modified trinucleotide d(TYT) (6) that
contained a cyanuric acid residue at the central position (1
AU260nm) was digested into nucleotides upon incubation for 2 h
at 37 °C with 5 EU (enzyme units) of nuclease P1 in a 30 mM
NaOAc and 0.1 mM ZnSO4 aqueous solution (pH 5.5) in a total
volume of 50 µL. Then, 10 EU of calf intestinal alkaline
phosphatase in 500 mM Tris and 1 mM EDTA (pH 8.5, 5 µL)
was added, and the resulting mixture was further incubated
for 1 h. The digestion mixture of 2′-deoxyribonucleosides was
then analyzed by HPLC onto a Hypercarb column. The elution
was performed with a 0 to 30% linear gradient of CH3CN in
0.25 mM ammonium formiate buffer over 40 min at a flow rate
of 0.5 mL/min (UV detection at 230 nm). The different collected
products were analyzed by ESI-MS in the negative mode.
(3) 1-{2-Deoxy-3-O-[2-cya n oeth oxy(d iisop r op yla m in o)-
p h osp h in o]-5-(4,4′-d im et h oxyt r it yl)-â-D-er yth r o-p en t ofu -
r a n osyl}cya n u r ic Acid (5). Compound 3 (0.145 mmol, 80 mg)
was coevaporated twice with dry pyridine and subsequently
dissolved in anhydrous CH2Cl2 (2 mL) under an argon atmo-
sphere. Diisopropylammonium tetrazolate (13 mg, 0.075 mmol)
and 2-cyanoethyl-N,N,N′,N′-tetraisopropyldiamidite (50 µL, 0.16
mmol) were added to the solution under stirring. The reaction
was monitored by TLC (95/5/1 CH2Cl2/MeOH/TEA). After 2 h
at room temperature, the mixture was diluted with ethyl acetate
(25 mL) and washed with a saturated NaHCO3 aqueous solution
(30 mL). The organic layer was dried over Na2SO4 and concen-
trated under vacuum. The resulting residue was purified by
flash chromatography on a silica gel column with a step gradient
of methanol (0 to 3%) in CHCl3/TEA (99/1, v/v) as the mobile
phase. Then, the collected fractions corresponding to the two
diastereomers were dried to afford compound 5 as a white foam
(yield of 68%, 0.098 mmol, or 73 mg): Rf ) 0.25 (95/5/1 CH2-
Cl2/MeOH/TEA); 31P NMR (101.21 MHz, CD3COCD3) δ 149.53
and 149.25 (two diastereoisomers); FAB-MS (negative mode) m/z
745.9 [M - H]-.
Sta bility Stu d ies of 1-(2-Deoxy-â-D-er yth r o-p en tofu r a n -
osyl)cya n u r ic Acid (1) u n d er th e Alk a lin e Con d ition s
Used for Ch em ica l Syn th esis of ODNs. Aqueous ammonia
(32%, 500 µL) was added to 0.5 AU230nm of compound 1 (the same
experiment was performed with the R-isomer 2) in sealed tubes.
The solutions were kept at either room temperature or 55 °C.
Then, the reactions were stopped at increasing time intervals
(0, 1, 2, 4, 8, 16, and 24 h) by freezing the samples in liquid
nitrogen and subsequent lyophilization. Samples were analyzed
by HPLC using a Hypercarb column. The elution was achieved
with a 0 to 10% linear gradient of acetonitrile in 25 mM
ammonium formiate buffer over the course of 30 min (flow rate
of 0.5 mL/min; UV detection at 230 nm).
En zym a tic Digestion of ODNs by 3′- or 5′-Exon u clea se
follow ed b y MALDI-TOF Ma ss Sp ect r om et r y An a lysis.
ODNs were precipitated twice in a 0.3 M ammonium acetate/
ethanol (1/3, v/v) solution prior to the enzymatic digestions.
(1) Digestion by Ca lf Sp leen P h osp h od iester a se (5′-
Exo). ODNs (0.1 AU260nm) were incubated at 37 °C with 10-3
EU of calf spleen phosphodiesterase (2 units/mL) in 0.02 M
ammonium citrate (pH 5) in a total volume of 30 µL. Aliquots
(1.5 µL) were withdrawn after increasing periods of time, and
the reactions were quenched by addition of 50 µL of water and
subsequent freezing of the samples in liquid nitrogen. Then, the
samples were lyophilized and analyzed by MALDI-TOF spec-
trometry following the conditions described above.
Sta bility Stu d ies of 1-(2-Deoxy-â-D-er yth r o-p en tofu r a n -
osyl)cya n u r ic Acid (1) u n d er th e Acid Con d ition s Used
for Ch em ica l Syn th esis of ODNs. A similar procedure, as
described above for the alkali stability assay, was used. This
involved incubation of compound 1 in an 80% acetic acid aqueous
solution for 0, 1, 2, 4, 8, 16, and 24 h at room temperature.
Sta bility Stu d ies of th e 1-(2-Deoxy-â-D-er yth r o-p en to-
fu r a n osyl)cya n u r ic Acid (1) u n d er Oxid izin g Con d ition s
Used for Ch em ica l Syn th esis of ODNs. Similarly, compound
1 was incubated in a 0.1 M oxidizing solution of iodine for 0, 1,
2, 4, 8, 16, and 24 h at room temperature.
Solid -P h a se Syn t h esis of Oligod eoxyr ib on u cleot id es.
The synthesis of cyanuric acid-containing oligodeoxyribonucle-
otides was performed at 1 µmol scale using an Applied Biosys-
tems Inc. 392 DNA synthesizer, with retention of the 5′-terminal
DMTr group (trityl-on mode). The standard 1 µmol DNA cycle
was used with a slight modification. This consisted in increasing
the duration of condensation by a factor of 4 for the modified
nucleoside phosphoramidite 5 (120 s instead of 30 s for normal
nucleoside phosphoramidites). Under these conditions, a cou-
pling efficiency of more than 90% for the modified monomer 5
was achieved.
Dep r otection a n d P u r ifica tion of Oligod eoxyr ibon u cle-
otid es. Upon completion of the synthesis, the alkali-labile
protecting groups of the oligodeoxyribonucleotides were removed
by treatment with concentrated aqueous ammonia (32%) at
room temperature for 4 h. Solvents were removed by evaporation
under vacuum. Then, the crude 5′-DMTr oligomers were purified
and deprotected on-line by reverse-phase HPLC using a poly-
meric support, as previously described (15). The modified 22-
mer ODN 10, used in repair and replication studies, was further
purified by preparative polyacrylamide gel electrophoresis and,
then, was desalted using a NAP-25 Sephadex column (Phar-
macia).
(2) Digestion by Sn a k e Ven om P h osp h od iester a se (3′-
Exo). ODNs (0.1 AU260nm) and 3 × 10-4 EU of snake venom
phosphodiesterase (3 units/mL) were added to 30 µL of 0.02 M
ammonium citrate (pH 9). The resulting solution was incubated
at 37 °C, and aliquots (1.5 µL) were withdrawn after increasing
periods of time. The reactions were quenched by addition of 50
µL of water and the samples frozen in liquid nitrogen. The
samples were lyophilized before being analyzed by MALDI-TOF
spectrometry following the conditions described above.
Th er m a l Den a tu r a tion Stu d ies. The melting experiments
were carried out using 1 nmol of ODN duplex. Thus, the
oligonucleotides [5′-d(ATC GTX ACT GAT CT)-3′] where X is G
or Y (0.125 and 0.118 AU260nm, respectively) and their comple-
mentary sequence [5′-d(AGA TCA GTC ACG AT)-3′] (0.125
AU260nm) were mixed in 20 µL of a buffer that contained 0.01 M
sodium phosphate, 0.1 M NaCl, and 1 mM EDTA (pH 7). The
DNA fragments were annealed by heating at 90 °C for 3 min
followed by slow cooling to 4 °C over the course of 3 h. The
hybridization solutions were diluted with 400 µL of buffer. Then,
UV absorbance measurements were taken in a 0.8 mL quartz
cell (0.2 cm path length) with a UV/vis spectrophotometer
equipped with a Peltier temperature controller. The absorbance
of the sample was monitored at 260 nm from 15 to 85 °C at a
heating rate of 1 °C/min. The reported data are the average of
three melting curves per ODN duplex.
DNA Rep a ir Stu d ies of Cya n u r ic Acid by F p g a n d
En d on u clea se III. The modified ODN 10 (10 pmol) was 5′-
end-labeled using [γ-32P]ATP and then purified using MicroSpin
G-25 columns. Nonlabeled modified ODN 10 (95 pmol) and the
complementary strand [5′-d(AGA TCA GTC ACG ATC CGA
AGT G)-3′] (150 pmol) were added to afford a double-stranded
DNA fragment that contained the lesion. The hybridization was
performed in 10 µL of buffer, consisting of 20 mM Tris-HCl, 1
mM EDTA, and 100 mM KCl (pH 7.5). This was achieved by
heating the resulting solution for 5 min at 80 °C and cooling
slowly to 4 °C. Water (40 µL) was added, and the solution was
P ip er id in e Tr ea tm en t of th e Cya n u r ic Acid -Con ta in in g
Oligod eoxyr ib on u cleot id es. Oligonucleotides were treated

Oligonucleotides Containing Cyanuric Acid Nucleoside
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 633
aliquoted (5 µL per reaction). Then, 5 µL of enzyme (Fpg or endo
III) at a final concentration of 2-100 ng/µL in a 2× buffer was
added to the aliquoted solutions of double-stranded DNA.
Reactions using increasing amounts of enzyme (2, 5, 10, 15, 25,
50, 75, and 100 ng/µL) were performed at 37 °C for 30 min and
stopped by addition of formamide (15 µL). Samples were
denatured by heating at 85 °C for 5 min and then electro-
phoresed on a 20% polyacrylamide-7 M urea gel at 1300 V for
30 min in TBE buffer [50 mM Tris, 50 mM boric acid, and 50
mM EDTA (pH 8)]. The reaction products were visualized by
autoradiography after exposure of the gel to X-ray films.
Mod ified ODN Rep lica tion Assa ys. Typically, 10 pmol of
modified DNA template 10 was annealed with 10 pmol of the
complementary 5′-32P-end-labeled 10-mer primer [5′-d(AGA TCA
GTC A)-3′] in 10 µL of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2,
1 mM DTT, and 0.2 mg of BSA buffer. The primer-template
solution was incubated at 80 °C for 5 min, and then cooled slowly
to 4 °C, at least, for 2 h. Subsequently, primer elongation by
the Klenow fragment was carried out at 25 °C in the same buffer
over the course of 1 h. Each sample contained 0.5 pmol of the
labeled primer-template duplex, 1 unit of polymerase, and the
four dNTPs (each at 100 µM) in a final volume of 25 µL. The
reactions were stopped by addition of formamide (15 µL).
Samples were denatured by heating at 85 °C for 5 min and then
electrophoresed on a 20% polyacrylamide-7 M urea gel at 1300
V for 30 min in TBE buffer. The reaction products were
visualized by exposing the gel to X-ray films. A similar experi-
ment using the unmodified 22-mer template [5′-d(CAC TTC
GGA TCG TGA CTG ATC T)-3′] was performed as a control.
Sch em e 1. F or m a tion of Cya n u r ic Acid Nu cleosid e
(1) fr om Ir r a d ia ted a n d P h otosen sitized DNA
Con stitu en ts
Resu lts a n d Discu ssion
Syn th esis of th e Modified P h osph or am idite Bu ild-
in g Block a n d Its In ser tion in to Defin ed Sequ en ce
Oligon u cleotid es. (1) Th e Cya n u r ic Acid Nu cleo-
sid e. Synthesis of 1-(2-deoxy-â-D-erythro-pentofuranosyl)-
cyanuric acid 1 (âdY) and its chemical incorporation into
ODNs have not been reported so far in the literature.
Our synthetic pathway (Scheme 2) for affording dY
involves the direct glycosylation of silylated cyanuric acid
with the 3,5-protected 2-deoxyribofuranoside chloride.
After removal of the toluoyl groups from the sugar moiety
and subsequent silica gel purification, an unresolvable
mixture of anomeric nucleosides 1 and 2 was obtained
in a 55% yield. The structure of 1 and 2 was assigned on
Sch em e 2a
1
the basis of UV, mass, and H and 13C NMR measure-
ments of the anomeric nucleoside mixture. This received
further confirmation by comparing the latter results with
the previously reported data for the â anomer prepared
by photosensitized oxidation (12). Thus, the UV spectrum
of dY (Figure 1) exhibits a lack of absorption at 260 nm
and a maximum, as a shoulder, at 220 nm. The mass
measurement, in the electrospray ionization mode, is
indicative of an empirical formula of C8H11O6N3. The two
1
anomers 1 and 2 show a major difference in the H NMR
features. This concerns the multiplicity of H-1′, which
appears as a doublet of doublets for the â-anomer 1 and
a triplet for the R-anomer 2. The results are in agrement
with the values already reported for modified pyrimidine
2′-deoxyribonucleosides exhibiting two carbonyl groups
in ortho positions with respect to the N-glycosidic bond
(16, 17). This mimics for both anomers a syn conforma-
tion since one of the two carbonyl group lies over the
sugar moiety. As a result, in both cases there was a shift
in the dynamic equilibrum between the C2′-endo and C3′-
endo furanose puckered forms toward the latter one. This
leads to a decrease in the trans J 1′,2′ coupling of the â
anomer, whereas an increase in the trans J 1′,2′′ coupling
of the R anomer is noted. Finally, NMR and reverse-phase
a
(a) 2,4,6-Trihydroxy-1,3,5-triazine (1 equiv), C4F9SO3K (2.4
equiv), HMDS (0.8 equiv), TMSCl (3.1 equiv), dry CH3CN, reflux,
16 h; (b) 1% NaOH, MeOH, 1 h, 55% 1 and 2; (c) DMTrCl (1.4
equiv), pyridine, 16 h, 40.5% 3; (d) 2-cyanoethyl-N,N,N′,N′-
tetraisopropyldiamidite (1.1 equiv), N,N′-diisopropylammonium
tetrazolate (0.5 equiv), CH2Cl2, 2 h, 68% 5.
HPLC analyses revealed that the â anomer was obtained
as the major product (1/2 R/â).
(2) P r ep a r a tion of Mod ified P h osp h or a m id ite
Bu ild in g Block s. The anomeric mixture of nucleosides
was then submitted to 5′-dimethoxytritylation and easily
resolved at this step by silica gel chromatography to
afford 3 (â anomer) as a pure product. The structure of

634 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Gasparutto et al.
F igu r e 1. UV spectrum, in water (pH 7), of dY.
Ta ble 1. Sequ en ces a n d Molecu la r Ma sses of th e
Mod ified Oligon u cleotid es Syn th esized a n d Used in Th is
Stu d ya
mass mass
calcd found
sequence (5′-3′)
length (Da) (Da)
6 T(Y)T
7 ATC T(Y)T ACT
8 ATC T(8-oxoGu a )T ACT
9 ATC GT(Y) ACT GAT CT
3
9
9
853 852.31
2667 2665.60
2704 2703.27
14 4232 4230.73
10 CAC TTC GGA TC(Y) TGA CTG ATC T 22 6679 6676.93
a
Y is cyanuric acid; 8-oxoGua is 8-oxo-7,8-dihydroguanine. All
the oligonucleotide masses have been obtained by ESI-MS mea-
surements in the negative mode.
both the 5′-DMTr-protected anomers (3 and 4) was
1
confirmed by H NMR analyses and ESI-MS measure-
ments. Then, the dimethoxytritylated â anomer 3 was
treated with the 2-cyanoethyl-N,N,N′,N′-tetraisopropyl-
diamidite phosphitylating reagent, affording the expected
phosphoramidite building block 5 in a good yield (68%)
after silica gel column chromatography.
F igu r e 2. PAGE analysis of the 5′-end-labeled 9-mer 5′-(ATC
TXT ACT)-3′ with X being Gua, 8-oxoGua, or Y, after treatment
with a 1 M piperidine aqueous solution at 90 °C for 0 and 30
min.
It should be noted that the stability of âdY under the
usual conditions of synthesis and deprotection of oligode-
oxyribonucleotides on solid support was demonstrated.
The ability to use 1 in standard automated ODN syn-
thesis was assessed by investigating its stability under
the various acid, alkali, and oxidizing conditions used
during the chemical preparation of DNA fragments.
Thus, âdY 1 was shown to be stable under the latter
conditions. Further studies performed on the R-anomer
2 confirmed the good stability of the cyanuric nucleoside
toward chemical degradation and anomerization.
(3) Syn th esis a n d Ch a r a cter iza tion of th e Oligo-
n u cleotid es. Several oligodeoxyribonucleotides (Table 1)
bearing a âdY residue were synthesized on solid support
using the phosphoramidite chemistry, with the modifica-
tions previously described in Materials and Methods.
This allowed a coupling efficiency of more than 90% for
the modified monomer. After ammonia deprotection at
room temperature for 4 h, the crude 5′-tritylated ODNs
were purified by RP-HPLC on polymeric support using
an on-line detritylation/purification procedure (15). The
purity and the homogeneity of the material were assessed
by several approaches, including analytical HPLC, poly-
acrylamide gel electrophoresis of 5′-32P-labeled fragments,
and mass spectrometry measurements. The molecular
masses of the oligonucleotides were inferred from elec-
trospray ionization mass spectrometry measurement in
the negative mode (Table 1). These results confirmed the
incorporation and the integrity of âdY 1 into the oligo-
mers.
modified oligonucleotides that contained the cyanuric
acid lesion allowed the determination of the stability of
1 under the piperidine conditions used to reveal alkali-
labile DNA modifications in oxidized oligonucleotides.
The stability experiment was extended to normal dGuo
and the 8-oxodGuo lesion incorporated into DNA. Then,
the comparative study of the stability was performed by
treating the 5′-32P-labeled oligonucleotides 5′-d(ATC TXT
ACT)-3′ where X is Gua, 8-oxoGua, or Y with piperidine
at 90 °C for 30 min. In a subsequent step, the resulting
DNA fragments were analyzed by denaturing polyacryl-
amide gel electrophoresis (Figure 2). It was found that
the cyanuric acid-containing oligonucleotide 7 was stable
under piperidine treatment. Thus, cyanuric acid cannot
be considered an alkali-labile site, as well as its oxidized
8-oxodGuo precursor which has been recently re-evaluted
as a piperidine-stable modification (11, 18-20).
Th er m a l Den a tu r a tion Stu d ies. To determine the
structural effect of the incorporation of cyanuric acid into
DNA, the thermal stability of the dY‚dC base pair was
evaluated. Thus, ODN 9, which contained a âdY lesion
at the central position, was annealed with its comple-
mentary DNA strand 5′-d(AGA TCA GTC ACG AT)-3′.
The melting temperature (Tm) of the duplex was deter-
mined by UV measurements at 260 nm. Interestingly,
the substitution of a dGuo residue with dY in the middle
of the sequence led to an important decrease in the Tm
of 15 °C (the Tm of the duplex that contained cyanuric
acid ) 40 ( 1 vs 55 ( 1 °C for the unmodified duplex).
The noticeable observed decrease in the melting temper-
ature suggests that the insertion of cyanuric acid sig-
nificantly induces a local destabilization of the hybrid
P ip er id in e St a b ilit y of Cya n u r ic Acid In ser t ed
in to Oligod eoxyr ibon u cleotid es. The preparation of

Oligonucleotides Containing Cyanuric Acid Nucleoside
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 635
F igu r e 3. RP-HPLC analysis of the enzymatic digestion
mixture of d(TGT) (A) and d(TYT) (B) treated with 5 EU of
nuclease P1 at 37 °C for 2 h and then with 10 EU of alkaline
F igu r e 4. MALDI mass spectra of the products resulting from
the digestion of modified oligonucleotide 9 by the 3′-exonuclease
after incubation for 2 (A) and 30 min (B).
phosphatase for
1 h (the chromatographic conditions are
reported in Materials and Methods). The detection of dY, which
exhibits a lack of absorption at 260 nm, was achieved at 230
nm (A230/260nm ) 11) upon injection of a large amount of
nucleosides arising from the digestion of 1 AU260nm of trinucle-
otide d(TYT). The inset shows the electrospray mass spectrum,
in the negative mode, of the collected dY residue.
analysis of both the digestion hydrolysates. This indicated
that the latter exonucleases failed to release the damaged
nucleoside from the oligonucleotidic chain (data not
shown). To confirm these results, enzymatic digestions
by SVPDE and CSPDE of the 14-mer modified ODN 9
were performed. Thus, the course of the hydrolysis of the
DNA strand by the nucleases was followed by withdraw-
ing aliquots from the digestion mixtures after increasing
periods of time. The DNA fragments were then analyzed
by MALDI-TOF mass spectrometry (21, 22). Using the
latter powerful technique, the different molecular ions
that were observed correspond to the digested DNA
fragments which differ in mass by successive loss of
nucleotides. The difference in mass between two adjacent
peaks allows us to identify the released nucleotide and
then to determine the overall sequence of the oligomer.
The unmodified 14-mer oligodeoxyribonucleotide 5′-
d(ATC GTG ACT GAT CT), used first as a control, was
totally hydrolyzed by both 5′- and 3′-exonucleases in less
than 15 min, allowing the determination of the complete
sequence (data not shown). In contrast, the presence of
nucleoside 1 in 9 induces a total resistance to digestion
by both SVPDE (3′-exo) and CSPDE (5′-exo). The mass
spectra after 2 and 30 min of hydrolysis by SVPDE are
shown in Figure 4. It was found that the enzyme induced
the release of the first height nucleotides at the 3′-end
of the oligonucleotide sequence (Figure 4A) but failed to
cleave the phosphodiester linkage between 1 and thymi-
dine, even after a prolonged treatment. This was inferred
from the observation of a single peak at m/z 1785.85 Da
(Figure 4B) that corresponds to the 6-mer [5′-d(ACT
GTY)-3′] (calcd mass of 1785 Da). On the other hand, the
digestion of ODN 9 by CSPDE for 30 min (Figure 5)
provided the 9-mer [5′-d(YAC TGA TCT)-3′] (calcd mass
of 2692 Da; found mass of 2691.82 Da). This shows that
the enzyme is able to digest sequentially the ODN from
the 5′-end until it reaches the cyanuric acid nucleoside
DNA structure, which may be mostly ascribed to a lack
of the hydrogen bonding with the complementary cytosine
base.
Nu clea se-Med ia ted Digestion s of Mod ified Oli-
gon u cleotid es Th a t Con ta in ed Cya n u r ic Acid . The
ability of several nucleases to cleave the phosphodiester
linkages between dY and the normal 2′-deoxyribonucleo-
sides was assessed using cyanuric acid nucleoside-
containing oligodeoxyribonucleotides.
First, the trimer d(TYT) was submitted to the action
of nuclease P1, followed by bacterial alkaline phos-
phatase. In addition, the unmodified trinucleotide d(TGT)
which was used as a reference was treated under the
same conditions. The resulting mixture of 2′-deoxyribo-
nucleosides arising from the digestion of d(TYT) was then
analyzed by reverse-phase HPLC (Figure 3B). Two HPLC
peaks, including those of thymidine (retention time of 28
min) and of an additional product (retention time of 10
min), were observed. The latter compound was collected
and then assigned as 1 by both HPLC co-injection with
an authentic sample of âdY and ESI-MS analysis. This
clearly shows the ability of nuclease P1 to cleave cyanuric
acid nucleoside from DNA. The latter observation pro-
vides additional support for the presence and the integ-
rity of 1 in the synthetic oligomers.
Similar enzymatic digestion experiments were per-
formed with the trinucleotide d(TYT) using exonucleases,
including snake venom phosphodiesterase (SVPDE, 3′-
exo) and calf spleen phosphodiesterase (CSPDE, 5′-exo),
in combination with bacterial akaline phosphatase. Sur-
prisingly, no peak corresponding to cyanuric acid nucleo-
side 1 was detected during the reverse-phase HPLC

636 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Gasparutto et al.
F igu r e 5. MALDI mass spectrum of the products resulting
from the digestion of modified oligonucleotide 9 by the 5′-
exonuclease after incubation for 30 min.
whose phosphodiester bond was found to resist to further
cleavage.
Rep a ir Assa ys of Cya n u r ic Acid -Con ta in in g Oli-
gon u cleotid es w ith F p g a n d En d on u clea se III P r o-
tein s. To investigate the biological significance of the
cyanuric acid base in oxidized DNA, attempts were made
to asses whether 1 may be a substrate for two base
excision repair enzymes, including formamidopyrimidine
DNA N-glycosylase (Fpg) and endonuclease III (endo III)
proteins. The E. coli Fpg protein is a well-known repair
enzyme which is able to excise several modified purine
bases from DNA duplexes, through both glycosylase
and AP-endonuclease activities (23, 24). Substrates rec-
ognized and excised by Fpg include 8-oxo-7,8-dihydro-
guanine (8-oxoG), 2,6-diamino-4-hydroxy-5-formamido-
pyrimidine (Fapy-guanine), 2,6-diamino-4-hydroxy-5-N-
methylformamidopyrimidine (Me-Fapy-guanine), and 4,6-
diamino-5-formamidopyridine (Fapy-adenine) (2, 4, 25-
28). It was also shown that 5-hydroxycytosine (5-OHC),
5,6-dihydrothymine (DHT), and N-3-(2-hydroxyisobutyric
acid)urea, three modified pyrimidine bases, are also
recognized and excised by Fpg (29-31).
On the other hand, previous studies have shown that
endonuclease III recognizes modified thymine and cy-
tosine bases, including 5,6-dihydrothymine (DHT), 5,6-
dihydroxy-5,6-dihydrothymine (thymine glycol), 5-hydroxy-
5,6-dihydrothymine, 5,6-dihydrouracil (DHU), 5,6-dihy-
droxy-5,6-dihydrouracil (uracil glycol), 5-hydroxy-5,6-
dihydrouracil, 5-hydroxyuracil (5-OHU), 6-hydroxy-5,6-
dihydrocytosine, 5-hydroxycytosine (5-OHC), urea, meth-
yltartronyl-N-urea, 5-hydroxy-5-methylhydantoin, and
alloxan (31-35).
Thus, to obtain further information about the substrate
specificity of both Fpg and endo III proteins, the modified
22-mer ODN 10 that contained dY at a central position
was incubated with the repair enzyme. This involved 5′-
32P-end labeling and subsequent hydridization of modi-
fied ODN 10 with its complementary sequence 5′-d(AGA
TCA GTC ACG ATC CGA AGT G)-3′, which contains a
cytosine in front of the cyanuric acid lesion. Then, the
damaged base excision by the repair enzymes was
determined by searching for the strand breakage of the
ODN using polyacrylamide gel electrophoresis. Using the
latter technique, it was shown that neither the Fpg
enzyme nor the endonuclease III protein is able to cleave
the modified DNA duplex at the site of 1 (data not
shown). Investigations are currently in progress to
determine the possible implication of bases other than
F igu r e 6. Primer extension reactions catalyzed by the Klenow
fragments. Using the modified 22-mer template [5′-d(CAC TTC
GGA TCY TGA CTG ATC T)-3′] primed with a 32P-labeled 10-
mer [5′-d(AGA TCA GTC A)-3′] (lane 1), primer extension
reactions were carried out by adding 1 unit of Klenow fragment
polymerase (lane 2, reference without nucleoside triphosphates)
in the presence of dNTP (lane 3), dATP (lane 4), dCTP (lane 5),
dGTP (lane 6), and dTTP (lane 7), as described in Materials
and Methods. Then, the reaction mixtures were subjected to
denaturing 20% PAGE, and the extended products were visual-
ized by exposing the gel to X-ray film.
cytosine in front of dY during the repair reaction and also
the excision of this lesion by other oxidized purine-specific
repair enzymes, namely, yeast and human Ogg1 proteins
(36-40).
In Vitr o Replication Exper im en ts with DNA P oly-
m er a se. The ability of the Klenow fragment to extend a
primer annealed with a template bearing a cyanuric acid
nucleoside was investigated. The primer was 32P-labeled
at its 5′-end so that extension by nucleotide incorporation
could be observed by sequencing polyacrylamide gel
electrophoresis (PAGE), the intensity of each band being
proportional to the number of molecules that terminates
the synthesis at a given position of the template. Figure
6 shows the denaturing PAGE bands obtained by elonga-
tion of the 10-mer primer 5′-d(AGA TCA GTC A)-3′ in
the presence of 5′-d(CAC TTC GGA TCY TGA CTG ATC
T)-3′ (template that contained a cyanuric acid residue in
the central position). Thus, when an equimolar concen-
tration of the four natural deoxyribonucleoside triphos-
phates was used in the reaction mixture (lane 3), it was
found that no chain termination opposite cyanuric acid
occurred during the extension of the primer. Indeed,
readthroughs giving rise to a full-length 22-mer product
were observed. This indicated that the cyanuric acid
residue does not block in vitro DNA synthesis.
To determine the nature of the base incorporated
opposite cyanuric acid, the same primer extension ex-
periments with Klenow polymerase were performed by
the “one-nucleotide extension assay”. This was achieved
by adding a single 2′-deoxyribonucleoside triphosphate
to each reaction mixture. The autoradiography (Figure
6, lanes 4-7) shows that dAMP was predominantly
incorporated opposite cyanuric acid (lane 4), dGMP being
also inserted to a lower extent (lane 6). These results
show that the cyanuric acid base exhibits an important

Oligonucleotides Containing Cyanuric Acid Nucleoside
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 637
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95, 288-293.
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into synthetic oligomers. Nucleosides Nucleotides 10, 755-761.
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DNA damage mediated by oxidation of an 8-oxoguanine residue.
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(1998) Gel electrophoretic detection of 7,8-dihydro-8-oxoguanine
and 7,8-dihydro-8-oxoadenine via oxidation by Ir(IV). Nucleic
Acids Res. 26, 2247-2249.
(12) Raoul, S., and Cadet, J . (1996) Photosensitized reaction of 8-oxo-
7,8-dihydro-2′-deoxyguanosine: identification of 1-(2-deoxy-â-D-
erythro-pentofuranosyl)cyanuric acid as the major singlet oxygen
oxidation product. J . Am. Chem. Soc. 118, 1892-1898.
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the 7,8-dihydro-8-oxoguanosine derivative. Formation of dioxetane
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6442.
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1151.
Con clu sion a n d P er sp ectives
The synthesis of cyanuric acid nucleoside (1) and its
incorporation into several oligonucleotides by the phos-
phoramidite approach were carried out for the first time.
The synthetic oligomers were isolated in good yields and
characterized by complementary techniques, showing the
integrity of the incorporated modified nucleoside. The
piperidine stability experiment performed with the modi-
fied oligonucleotides supported the high stability of the
cyanuric acid lesion inserted into DNA strands. A sig-
nificant decrease in the melting temperature of a double-
stranded 14-mer DNA fragment that contained a Y‚C
base pair was observed. This suggests that the presence
of cyanuric acid induces a local destabilization of the
duplex DNA structure. The processing of 1 by different
nucleases was studied. Thus, it was shown that nuclease
P1 is able to quantitatively cleave the cyanuric acid
residue from the oligonucleotides while both SVPDE and
CSPDE failed to release the latter modification from the
DNA fragment. On the other hand, the ability of two
repair enzymes, namely, the Fpg and the endonuclease
III proteins, to excise cyanuric acid was investigated. A
series of experiments was performed which clearly indi-
cated that cyanuric acid was not the substrate for either
repair enzyme. The biochemical study was then extended
to the evaluation of the mutagenic properties of the
cyanuric acid lesion. This involved the determination of
the base-specific incorporation directed by this residue
during in vitro replication using the Klenow fragment.
Thus, 1 does not block the polymerase which incorporates
adenine opposite the damage, guanine being also slighty
inserted.
The cyanuric acid nucleoside and the different modified
ODNs prepared in this work are actually used as tools
for investigations aimed at determining the mechanism
and the level of formation of 1 in single- and double-
stranded oxidized DNA.
Ack n ow led gm en t. The contributions of Colette Le-
brun (LRI/SCIB/CEA-Grenoble, Grenoble, France) and
Daniel Ruffieux (Biochimie C/CHRU-Grenoble, Grenoble,
France) to the FAB and electrospray mass measurements
are gratefully acknowledged. We are indebted to Dr.
Serge Boiteux for the gift of endonuclease III and Fpg
proteins. Financial support from the Comite´ de Radio-
protection (Electricite´ de France) is acknowledged.
(20) Gasparutto, D., Ravanat, J .-L., Ge´rot, O., and Cadet, J . (1998)
Characterization and chemical stability of photooxidized oligo-
nucleotides that contain 2,2-diamino-4-[(2-deoxy-â-D-erythro-
pentofuranosyl)amino]-5(2H)-oxazolone. J . Am. Chem. Soc. 120,
10283-10286.
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assisted laser desorption ionization time-of-flight mass spectrom-
etry: a powerful tool for the mass and sequence analysis of
natural and modified oligonucleotides. Nucleic Acids Res. 21,
3191-3196.
(22) Smirnov, I. P., Roskey, M. T., J uhasz, P., Takach, E. J ., Martin,
S. A., and Haff, L. A. (1996) Sequencing oligonucleotides by
exonuclease digestion and delayed extraction matrix-assisted
laser desorption ionization time-of-flight mass spectrometry. Anal.
Biochem. 238, 19-25.
(23) Boiteux, S., O’Connor, T. R., and Laval, J . (1987) Formamidopy-
rimidine-DNA glycosylase of Escherichia coli: cloning and se-
quencing of the Fpg structural gene and overproduction of the
protein. EMBO J . 6, 3177-3183.
(24) Boiteux, S., O’Connor, T. R., Lederer, F., Gouyette, A., and Laval,
J . (1990) Homogeneous Fpg protein of Escherichia coli: a DNA
glycosylase which excises imidazole ring-opened purines and nicks
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